DNA vaccine against multitypes of avian influenza viruses and influenza virus-like particles comprising adjuvant-fused M2 protein

ABSTRACT

A DNA vaccine comprising hyperglycosylated mutant HA gene, which is derived from avian influenza virus, is provided. A DNA vaccine composition comprising: (a) the DNA vaccine; and (b) a booster is also provided. An influenza virus-like particle comprising adjuvant-fused M2 protein is further provided. A method for eliciting an immune response against a plurality of avian influenza virus subtypes in a subject, comprising delivering the DNA vaccine or the DNA vaccine composition to tissue of the subject is also provided.

FIELD OF THE INVENTION

The present invention relates to a DNA vaccine. More specifically, thepresent invention relates to a DNA vaccine comprising hyperglycosylatedantigen. The present invention also relates to a DNA vaccine compositionand a method for eliciting an immune response against multiple avianinfluenza virus subtypes in a subject using the same. The inventionfurther relates to an influenza virus-like particle comprisingadjuvant-fused M2 protein.

BACKGROUND OF THE INVENTION

Highly pathogenic avian influenza (HPAI) H5N1 viruses and their capacityfor transmission from birds to humans have raised worldwide concernsabout a potential forthcoming human pandemic. With the continued spreadof H5N1 influenza virus, new virus strains have emerged and willcontinue to change and evolve in the future. The World HealthOrganization has classified the H5N1 viruses isolated recently into 10clades (or sublineages) based on the phylogenetic analysis of viralhemagglutinin (HA) sequences of H5N1 viruses. With the continuous threatof a new influenza pandemic arising from avian reservoirs, thedevelopment of broadly protective vaccines is particularly important. Todate, the broadly protective H5N1 vaccines have been mainly achievedusing novel adjuvant formulations.

However, the inherent nature of influenza virus antigenic changes hasnot been taken into accounts in the immunogen designs for developingbroadly protective H5N1 vaccines. Refocusing antibody responses havebeen proposed by designing the immunogens that can preserve the overallfold of the immunogen structure but selectively mutate the “undesired”antigenic sites that are highly variable (escape mutants evadeprotective immune responses), immunosuppressive (downregulate the immuneresponse to the infection), cross-reactive (the immune response inducesa reaction to a protein resembling the immunogen). The immunogen designby refocusing antibody responses has been applied for HIV-1 vaccinesusing the hyperglycosylated HIV-1 gp120 immunogens where the undesiredeptiopes are masked by selective incorporations of N-linked glycans. Theglycan masking strategy has been also recently reported to designinfluenza virus vaccines that can enhance the antibody responses againsta broad range of H3N2 intertypic viruses. However, there is no reportfor the use of glycan-masking immunogen design for H5N1 vaccines.

DNA vaccine has been considered as the revolutionary vaccinology withthe advantages in offering genetically antigen design, time tomanufacturing, long stability without the need for cold chains supply,and the immunogenicity predominantly elicited by T cells through theendogenerous antigen processing pathways. However, the apparent lowimmunogenicity of DNA vaccines in large animals (including humans) hasbeen overcome using novel delivery systems such as gene-guns orelectroporation. Additionally, the DNA vaccine-elicited immune responsescan be further augmented using the heterologous prime-boost immunizationregimen where the booster dose uses a different vaccine formatcontaining the same or similar antigens. Examples of DNA vaccineprime-boost immunization strategy has been reported for the inactivatedinfluenza virus, live-attenuated influenza virus, recombinantadenovirus, virus-like particles (VLPs) and recombinant subunit proteinsin adjuvants. Furthermore, human vaccines receiving the H5 DNA vaccinepriming followed by a booster with inactivated H5N1 vaccine were foundto enhance the protective antibody responses (HAI) and in some casesinduce the haemagglutinin-stem-specific neutralizing antibodies.

Influenza VLPs are noninfectious and have a size and morphology that aresimilar to those of native virion structures, but they do not containthe genomic RNAs for virus replication. The assembly of influenza VLPsdepends on the interactions of M1 proteins and/or other viral surfaceproteins, such as HA, NA, and M2, with the cellular lipid membranes. Theinteractions of M1 protein with the cytoplasmic tails of HA and NAspikes can increase the lipid membrane binding of M1 proteins inassembling influenza virus. The interactions of HA and NA with the M1protein can also reduce the formation of elongated intracellularimmature particles and improve the secretion of spherical mature VLPs.Additionally, the cytoplasmic tails of M2 protein, by interacting withthe M1 protein, further promote the budding and release of the influenzavirions. Recently, the M2 protein was found to act as the plasmamembrane-targeting signal for the budding and egress of influenzavirions. Host cell proteins can be recruited into the VLPs, as recentlyshown by LC/MS/MS analyses. Therefore, the biosynthesis of influenzaVLPs is a self-assembly process that involves complex interactions ofviral and cellular components.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention can be more fully understood by reading the subsequentdetailed descriptions and examples with references made to theaccompanying drawing, wherein:

FIG. 1 shows expression and characterization of DNA-HA and FliC-VLP. (A)The cell lysates of 293A cells transfected with either DNA-HA or emptyvector were treated with Endo H, PNGase F and Trypsin, and analyzed byWestern blots. Full-length HA proteins showed the presence of amolecular weight of approximately 75 kDa and HA1 proteins showed thepresence of a molecular weight of about 46 kDa. (B) FliC-VLPs werepurified by sucrose gradient sedimentation and the results showed thefractions 6 to 10 from the sucrose density gradient contained all fourproteins. (C) Electron microscopic visualization demonstrated thespherical morphology of the FliC-VLPs with a particle size around 100nm.

FIG. 2 shows total anti-HA IgG titers elicited by DNA-HA and FliC-VLP.Asterisks indicate a statistically significant difference (p<0.05).

FIG. 3 shows neutralizing activities of the sera from immunized mice bythe (A) HI and (B) NT titers against the NIBRG-14 (clade 1) H5N1influenza virus. For calculation purposes, an undetectable level wasscored as a titer equal to one. Individual titer (points) and geomean(lines) was given for each group.

FIG. 4 shows analytical result of amino acid variation in the HA of 163avian influenza virus strains. Eleven amino acids in the HA1 subunit,including the 83, 86, 94, 124, 129, 138, 140, 155, 162, 189 and 252residues were calculated to have relatively higher scoring numbers.

FIG. 5 shows nine N-linked glycosylation sites: 83NNT (SEQ ID NO:4),86NNT (SEQ ID NO:6), 94NFT (SEQ ID NO:8), 127NSS (SEQ ID NO:10), 138NRT(SEQ ID NO:12), 140NSS (SEQ ID NO:14), 161NRS (SEQ ID NO:16), 182NDT(SEQ ID NO:18), and 252NAT (SEQ ID NO:20). Underlined triplet aminoacids and arrows point away from wild-type sequence to amino acid changethat resulted in N-linked glycosylation sequence.

FIG. 6 shows the results of hemadsorption assay. (A) Positive control;(B) negative control; (C) 83NNT; (D) 86NNT; (E) 94NFT; (F) 127NSS; (G)138NRT; (H) 161NRS; (I) 182NDT; and (J) 252NAT.

FIG. 7 shows characterization of hyperglycosylated HA. The six HA mutantproteins (83NNT, 86NNT, 94NFT, 127NSS, 138NRT, 161NRS) with N-linkedglycans addition were illustrated by the increased molecular weights andreduced to the same molecular weight after PNGase F treatment.

FIG. 8 shows total anti-HA IgG titers elicited by hyperglycosylated HA.Individual titer (points) and geomean (lines) was given for each group.

FIG. 9 shows neutralizing activities of sera from immunized mice by the(A) HI and (B) NT titers against the NIBRG-14 (clade 1) H5N1 influenzavirus. For calculation purposes, an undetectable level was scored as atiter equal to one. Individual titer (points) and geomean (lines) wasgiven for each group. Asterisks indicate a statistically significantdifference (p<0.05).

FIG. 10 shows neutralizing activities of sera from immunized mice by the(A) HI and (B) NT titers against the Mongolia/2/2006 (clade 2.2) H5N1influenza virus. For calculation purposes, an undetectable level wasscored as a titer equal to one. Individual titer (points) and geomean(lines) was given for each group. Asterisks indicate a statisticallysignificant difference (p<0.05).

FIG. 11 shows construction of baculovirus expression vector forinfluenza VLP production. Influenza VLPs are obtained from Sf9 cellsthat are infected with (A) a single baculovirus that encodes two viralproteins (BacHA-M1) (B) two baculoviruses that encode three viralproteins (BacHA-M1 and BacNA) (C) two baculoviruses that encode fourviral proteins (BacHA-M1 and BacNA-M2). pH: polyhedron promoter; p10:p10 promoter.

FIG. 12 shows sucrose gradient analyses of the influenza VLPs obtainedby the expression by baculovirus of (A) two viral proteins (HA and M1);(B) three viral proteins (HA, NA, M1); and (C) four viral proteins (HA,NA, M1, M2). Purified sucrose fractions were resolved in SDS-PAGE gelsand reacted with anti-HA, anti-M1, anti-NA, and anti-M2 antibodies.

FIG. 13 shows TEM analyses of influenza VLPs expressed by baculovirususing (A-D) two viral proteins (HA and M1); (E-H) three viral proteins(HA, NA, M1); and (1-L) four viral proteins (HA, NA, M1, M2). The TEMimages present quadruple samples for each case of negative staining ofinfluenza VLPs with uranyl acetate.

FIG. 14 shows production of influenza VLPs with EGFP/M2 fusion protein.(A) Sucrose gradient analysis of influenza VLPs, reacted with anti-HA,anti-NA, anti-M1, anti-EGFP specific antibodies; (B-E) TEM images ofinfluenza EGFP-VLPs that are negatively stained with uranyl acetate,showing quadruple samples.

FIG. 15 shows EGFP-VLPs in A549 cells visualized by confocalfluorescence microscopy. A549 cells were labeled with DiD and EGFP-VLPswere labeled with DiI. (A) Excitation by 488 nm line from laser and 633nm line from laser; (B) excitation by 561 nm line from laser and 633 nmline from laser.

FIG. 16 shows production of influenza VLPs with FliC/M2 fusion protein.(A) Sucrose gradient analysis of influenza VLPs reacted with anti-HA,anti-NA, anti-M1, anti-M2 specific antibodies; (B-E) TEM images ofinfluenza FliC-VLPs that are negatively stained with uranyl acetate,showing quadruple samples.

FIG. 17 shows production of influenza VLPs with PRO/M2 fusion protein.(A) Sucrose gradient analysis of the influenza VLPs reacted withanti-HA, anti-NA, anti-M1, and anti-M2 specific antibodies; and (B) TEMimages of influenza PRO-VLPs that are negatively stained with uranylacetate, showing quadruple samples.

FIG. 18 shows intracellular TNF-α production of BMDCs treated with (A)non-fabricated VLPs, (B) FliC-VLPs, (C) PRO-VLPs, (D) PBS (negativecontrol), or (E) 20 ng/mL LPS (positive control). TNF-α production wasdetected by FACS analysis in groups of treated (black lines) anduntreated (gray lines) BMDCs. Average TNF-α+BMDCs of gated M1 wereobtained from at least three independent experiments.

FIG. 19 shows analytic results of CD40 and CD86 surface markers on BMDCstreated with non-fabricated VLPs, FliC-VLPs and PRO-VLPs. The meanfluorescence intensity (MFI) of the groups of treated (black lines) anduntreated (gray lines) BMDCs are presented in (A) CD40⁺CD11c⁺ and (B)CD86⁺CD11c⁺ phenotypes. Results are obtained from triplicateexperiments.

FIG. 20 shows neutralization of antisera collected from mice immunizedwith VLPs, FliC-VLPs and PRO-VLPs using H5pp of (A) the homologous KAN-1strain and (B) the heterologous Anhui strain.

SUMMARY OF THE INVENTION

The present invention relates to a DNA vaccine comprisinghyperglycosylated HA gene(s), which is derived from avian influenzavirus, wherein the mutant HA gene encodes a protein having a mutation atamino acid residue selecting from the group consisting of 83, 86, 94,127, 138, 161, 182, and 252. The present invention also relates to a DNAvaccine composition comprising: (a) an above-mentioned DNA vaccine; and(b) a booster. The present invention further relates to an influenza VLPcomprising adjuvant-fused M2 protein.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “wild-type” refers to a naturally occurringorganism. The term also relates to nucleic acids and proteins found in anaturally occurring organism of a naturally occurring population arisingfrom natural processes, such as seen in polymorphisms arising fromnatural mutation and maintained by genetic drift, natural selection andso on, and does not include a nucleic acid or protein with a sequenceobtained by, for example, recombinant means.

“Immunogen” and “antigen” are used interchangeably herein as a moleculethat elicits a specific immune response of antibody (humoral-mediated)and/or T cell origin (cell-mediated), for example, containing anantibody that binds to that molecule or a CD4⁺ or CD8⁺ T cell thatrecognizes a virally-infected cell expressing that molecule. Thatmolecule can contain one or more sites to which a specific antibody or Tcell binds. As known in the art, such sites are known as epitopes ordeterminants. An antigen can be polypeptide, polynucleotide,polysaccharide, a lipid and so on, as well as a combination thereof,such as a glycoprotein or a lipoprotein. An immunogenic compound orproduct, or an antigenic compound or product is one which elicits aspecific immune response, which can be humoral, cellular or both.

An “individual” or “subject” or “animal”, as used herein, refers tovertebrates that support a negative strand RNA virus infection,specifically influenza virus infection, including, but not limited to,birds (such as water fowl and chickens) and members of the mammalianspecies, such as canine, feline, lupine, mustela, rodent (racine, andmurine, etc.), equine, bovine, ovine, caprine, porcine species, andprimates, the latter including humans.

As used herein, the term “a plurality of” is employed to describe thenumber of elements and components of the present invention. Thisdescription should be read to more than one unless it is obvious that itis meant otherwise.

As used herein, the term “a” or “an” is employed to describe elementsand components of the invention. This is done merely for convenience andto give a general sense of the invention. This description should beread to include one or at least one and the singular also includes theplural unless it is obvious that it is meant otherwise.

As used herein, the term “or” is employed to describe “and/or”.

Accordingly, the present invention provides a DNA vaccine comprisinghyperglycosylated HA gene(s), which is derived from avian influenzavirus, wherein the mutant HA gene encodes a protein having one or moremutations at amino acid residue selecting from the group consisting of83, 86, 94, 127, 138, 161, 182, 252, and the combination thereof.

In one embodiment, the hyperglycosylated HA gene encodes a proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, and 20. In another embodiment,the mutant HA gene encodes a protein comprising an amino acid sequenceof SEQ ID NOs: 4, 6, or 10.

In one embodiment, delivery of the DNA vaccine into a subject elicits animmune response against a plurality of avian influenza virus subtypes inthe subject. In another embodiment, the delivery is achieved by way of,for example but not limited to, subcutaneous injection, intramuscularinjection, oral administration, spraying or gene gun injection.

The present invention also provides a DNA vaccine compositioncomprising: (a) an above-mentioned DNA vaccine; and (b) a booster.

In one embodiment, the booster is an influenza VLP. In anotherembodiment, the influenza VLP is derived from cell infected byrecombinant baculoviruses comprise one or more plasmids containing HAgene, M1 gene, NA gene and FliC-M2 gene, which encodes FliC-M2 fusionprotein.

In one embodiment, the DNA vaccine composition further comprises anadjuvant. In another embodiment, the adjuvant is an aluminum-containingadjuvant.

In one embodiment, the DNA vaccine and the booster have a mass ratio inthe range of 1:2 to 17:6. In another embodiment, the DNA vaccine and thebooster have a mass ratio in the range of 5:6 to 5:2. In still anotherembodiment, the DNA vaccine and the booster have a mass ratio of 5 to 3.

In one embodiment, delivery of the DNA vaccine composition into asubject elicits an immune response against a plurality of avianinfluenza virus subtypes in the subject. In another embodiment, thedelivery is achieved by way of, for example but not limited to,subcutaneous injection, intramuscular injection, oral administration,spraying or gene gun injection.

The present invention further provides an influenza VLP comprisingadjuvant-fused M2 protein. In one embodiment, the influenza VLP furthercomprises HA protein, NA protein and M1 protein. In another embodiment,the adjuvant is flagellin (FliC) or profiling (PRO).

The next examples provide some exemplary embodiments of the presentinvention as follows:

EXAMPLES

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

Example 1 Material and Methods

Construction of DNA-HA Vaccine Vector

The cDNA of the HA gene of influenza virus A/Thailand/1(KAN-1)/2004/H5N1(clade 1), SEQ ID NO: 1, was provided by Prasert Auewarakul, SirirajHospital, Thailand. The full-length HA sequence was inserted into apcDNA™3.1(+) vector (Invitrogen) using KpnI/NotI cut site. Theconstructed plasmid containing H5HA was transfected into 293A cells byusing Turbofect reagent (Fermentas). Following transfection for 48hours, the cell lysates were collected by centrifugation at 5000 rpm for10 minutes and HA expression was analyzed by Western blotting withanti-H5HA antibodies (ab21297; Abcam).

HA Glycosylation Pattern and Trypsin Treatment

For characterizing the HA glycosylation pattern, 293A cells wereharvested after transfected with DNA-HA vectors for 48 hours. The celllysates were treated with EndoH or PNGase F for 2 hours at 37° C., andthe H5HA glycosylation pattern was determined by Western blotting. Fortrypsin treatment, the cell lysates were incubated with trypsin for 30minutes on ice, and the cleavage of HA0 into HA1 and HA2 was observed byWestern blotting.

Preparation of VLPs

VLPs were prepared as described previously (Wei H J et al., Vaccine 29(2011): 7163-7172). Briefly, HA (SEQ ID NO: 1) and M1 (SEQ ID NO: 21)were cloned into a pFastBac™ Dual vector (Invitrogen), while NA (SEQ IDNO: 27) and FliC-M2 (SEQ ID NO: 25), expressing FliC-M2 fusion proteins,were cloned into the other one to produce the recombinant baculoviruses.Sf9 cells co-infected with recombinant baculoviruses were harvested at72 hours post-infection, and supernatants containing FliC-VLPs wereconcentrated by filtration with a 500 kDa filter membrane. Theconcentrate were loaded on 0-60% sucrose gradients and centrifuged for 4hours at 33,000 rpm. The desired particles were observed by Westernblotting using anti-H5HA antibodies (ab21297; Abcam), anti-NA antibodies(ab70759; Abcam), anti-M1 antibodies (ab25918; Abcam), and anti-M2antibodies (NB100-2073; Novus). The particles were also confirmed bytransmission electron microscopy (TEM) as described previously (Wei H Jet al., Vaccine 29 (2011): 7163-7172).

Preparation of Hyperglycosylated H5HA

Mutations were introduced into the HA gene by using the site-directedmutagenesis, and plasmids encoding wild-type H5HA gene (SEQ ID NO: 1)were used as templates. The 50 μL PCR reaction was carried out with 100ng templates, 2 mM primer pair, 200 mM dNTPs and 2 U of DNA polymerase.The PCR products were purified and further treated with DpnI for 2 hoursat 37° C. DpnI treated products were transformed into TOP10 competentcell and then the mutated plasmids were isolated.

Hemadsorption Assay

293A cells were transfected with wild-type and mutated H5HA DNA vectors,and the cells were harvested at 72 hours post infection. Followingphosphate-buffered saline (PBS) wash, sufficient 0.5% turkey red bloodcells (RBCs) were added to cover cell monolayer and incubate for 30minutes. Adsorption of RBCs on the transfected cells was observed afterrinse with PBS two times.

Mouse Immunization

6 to 8 weeks old female BALB/c mice were immunized with heterologousprime-boost strategy by 50 μg of DNA and 30 μg of purified VLPs mixedwith Alum adjuvant in PBS. Immunizations were performed at weeks 0, 3 byintramuscular injection. Blood was collected at 14 days followingimmunization, and serum was isolated. Serum samples were inactivated at56° C. for 30 minutes and stored in −20° C. All experiments wereconducted in accordance with the guidelines of the Laboratory AnimalCenter of National Tsing Hua University (NTHU). Animal use protocolswere reviewed and approved by the NTHU Institutional Animal Care and UseCommittee (approval no. 09733).

Enzyme-Linked Immunosorbent (ELISA) Assay

ELISA assay was performed as described previously (Lin S C et al., PLoSOne 6 (2011): e20052). Briefly, 2 μg/mL of purified protein were coatedon 96 well plates and then blocked with BSA. Serial dilutions of eachserum sample were incubated in the plates for 1 hour and removed by 3times wash. Goat anti-mouse IgG conjugated HRP (Bethyl Laboratories,Inc.) was incubated in the plates for 1 hour followed by 3 times wash.After the reaction with TMB substrate stop, plates were read at 450 nmabsorbance. End-point titer was determined as the reciprocal of thefinal dilution giving an optical of two-fold absorbance of negativecontrol.

Hemagglutinin Inhibition (HI) and Neutralization (NT) Assays

HI and NT assays were performed as described previously (Huang M H etal., PLoS One 5 (2010): e12279). For HI assay, serum samples (two-folddilutions starting with an initial dilution of 1:10) were incubated withfour HA units of influenza strain. Turkey RBCs were then added and theinhibition of agglutination was scored. The serum titer was expressed asthe reciprocal of the highest dilution that showed complete inhibitionof HA. For NT assay, the 200 TCID₅₀ per well of virus were incubatedwith two-fold-diluted mice sera at a starting dilution of 1:40. Mixturesof virus and serum were transferred to monolayers of MDCK cells andincubated for 4 days. The neutralizing titer was defined as thereciprocal of the highest serum dilution at which the infectivity of theH5N1 virus was neutralized in 50% of the wells. Infectivity wasidentified by the presence of cytopathy on Day 4 and the titer wascalculated using the Reed-Muench method.

Statistic Analysis

All results were analyzed using two-tailed Student's t tests, with a Pvalue of <0.05 indicating statistical significance

Results

Construction and Characterization of DNA-HA Vaccine Vector and FliC-VLPsfor Prime-Boost Immunization

The DNA vaccine vector (DNA-HA) encoding the full-length cDNA of theA/Thailand/1 (KAN-1)/2004/H5N1 (clade 1) HA gene (SEQ ID NO: 1) wasconstructed from the pcDNA™3.1(+) vector. Expression of the full-lengthHA protein was demonstrated in 293A cells transfected with the DNA-HAvector and analyzed in Western blots to show the presence of a molecularweight of approximately 75 kDa (FIG. 1A). The expressed HA in 293A cellswas sensitive to PNGase F treatment but resistant to EndoH digestion,suggesting as a glycoprotein containing complex type N-linked glycanprofiling (FIG. 1A). The expressed HA in DNA-HA transfected 293A cellswas also sensitive to trypsin treatment by cleavage from HA0 to HA1 andHA2 subunits, as shown the presence of HA1 at a molecular weight about46 kDa (FIG. 1A).

The FliC-containing VLPs (FliC-VLPs) were obtained from Sf9 cellsinfected with two recombinant baculoviruses encoding four of theinfluenza virus genes of HA, NA, and M1, and the fusion of M2 and theSamollena fliC genes (Wei H J et al., Vaccine 29 (2011): 7163-7172).FliC-VLPs were obtained from the culture supernatants ofbaculovirus-infected Sf9 cells, purified by ultracentrifugation andsucrose gradient sedimentation. The results show the fractions 6 to 10from the sucrose density gradient contained all four viral or fusionproteins (FIG. 1B). Electron microscopic visualization demonstrated thespherical morphology of the FliC-VLPs with a particle size around 100 nm(FIG. 1C).

To investigate the combined use of DNA-HA vaccine vector and FliC-VLPfor prime-boost immunization studies, BALB/c mice were immunizedintramuscularly (i.m) for two doses within a three-week interval as thefollowing prime-boost regimens: (i) PBS+PBS (ii) FliC-VLP+FliC-VLP (iii)DNA-HA+DNA-HA (iv) DNA-HA+FliC-VLP. Sera were collected at two weeksafter the second dose in immunized mice. The results show that theHA-specific total IgG titer by DNA-HA vaccine vector priming, followedby FliC-VLP boosting was significantly higher than two-dose immunizationusing DNA-HA vector and FliC-VLPs (FIG. 2). Neutralizing activitiesrevealed by measuring the HI and NT titers against the NIBRG-14(clade 1) H5N1 influenza virus show that the DNA-HA vector priming andFliC-VLP boosting regiment elicited the highest magnitude ofneutralizing antibodies in mice (FIGS. 3A-B).

Design of Hyperglycosylated HA Based on Amino Acid Sequences of H5N1Human Isolates

To design the hyperglycosyalted HA DNA vaccines, sequence alignmentanalysis was first conducted from 163 HPAI H5N1 human isolates(sequences retrieved from NCBI Database). The amino acid differences inthese HA1 protein sequences were analyzed based on the following scoringnumbers, 4 (different amino acid), 2 (weak similar amino acid), 1(strong similar amino acid), 0 (identical amino acid) as characterizedby the Vector NTI Similar Tables. According to the alignment plot shownin FIG. 4, eleven amino acid residues in the HA1 protein were identifiedto have a relatively higher scoring numbers, including the 83, 86, 94,124, 129, 138, 140, 155, 162, 189, and 252 residue. To design theantibody-refocused immunogens, site-directed mutagenesis is conducted ineach of the five regions with mutations to allow the addition of theN—X—S/T motif (for N-linked glycosylation site) but avoid the receptorbinding sites (Yang Z Y et al., Science 317 (2007): 825-828; and Yang Het al., PLoS Pathog 6 (2010): e1001081).

Nine N-X-S/T motifs were thus introduced into HA1, including 83NNT(99-101 of the SEQ ID NO: 4), 86NNT (102-104 of the SEQ ID NO: 6), 94NFT(110-112 of the SEQ ID NO: 8), 127NSS (143-145 of the SEQ ID NO: 10),138NRT (154-156 of the SEQ ID NO: 12), 140NSS (156-158 of the SEQ IDNO:14), 161NRS (177-179 of the SEQ ID NO: 16), 182NDT (198-200 of theSEQ ID NO: 18), and 252 NAT (268-270 of the SEQ ID NO: 20) (FIG. 5),wherein 83NNT, 86NNT, 94NFT, 127NSS, 138NRT, 140NSS, 161NRS, 182NDT and252NAT are amino sequences of the mature protein whereas SEQ ID NOs: 4,6, 8, 10, 12, 14, 16, 18, and 20 are amino sequences of the immatureprotein. Each of the refocusing hyperglycosylated HA genes containingthe specified N-linked glycosylation sites were cloned into the DNA-HAvaccine vector. However, only six out the nine immunofocusing HAretained the hemagglutination property for Turkey red blood cells aftertransfection into 293A cells (FIG. 6). The six HA mutant genes (83NNT,86NNT, 94NFT, 127NSS, 138NRT and 161NRS) were also investigated for theintroduction of N-linked glycans in the HA antigens as illustrated bythe increased molecular weights and reduced to the same molecular weightafter PNGase F treatment (FIG. 7).

Priming with Hyperglycosylated HA DNA Vaccines Followed by FliC-VLPBoosting

To investigate the antibody responses elicited by these sixhyperglycosylated HA mutants (83NNT, 86NNT, 94NFT, 127NSS, 138NRT and161NRS), mice were immunized with each DNA-HA vector twice followed witha third boosting dose with FliC-VLPs on a three-week interval. Theresults show that no significant differences of the HA-specific totalIgG titers of all the immunized groups with the hyperglycosyalted HA DNAvaccines compared to the wild-type control (FIG. 8). The 83NNT and 86NNTHA mutants elicited higher HI titers (FIG. 9A) but only the 83NNT HAmutant had higher NT titer (FIG. 9B) against the NIBRG-14 virus thatbelongs to the same H5N1 clade 1 strain. The HI and NT titers of thesesera against the Mongolia/2/2006 H5N1 virus of the clade 2.2 strain werealso measured. The data presenting as cross-clade functional antibodiesshow that the 83NNT, 86NNT, 127NSS HA mutants elicited higher HI titers(FIG. 10A) and the 83NNT, 86NNT, 127NSS, 161 NRS HA mutants had higherNT titers (FIG. 10B). Taken together, the 83NNT mutant can elicit morepotent HI and NT titers against both the NIBRG-14 (clade 1) andMongolia/2/2006 (clade 2.2) HPAI H5N1 viruses.

Example 2 Methods and Materials

Cell Lines

Sf9 cells (ATCC CRL-1711) (Invitrogen) were derived from pupal ovariantissue of the fall armyworm, Spodoptera frugiperda. Sf9 cells weremaintained in T-flasks at 28° C. with SF-900II serum free medium (GIBCO)that contained 100 units/mL penicillin and 100 μg/mL streptomycin(Invitrogen). For suspension cultures, Sf9 cells were inoculated in 500mL spinner flasks (Belleco) at 60 rpm at 27° C. with 300 mL of the samemedium. A549 cells (human lung carcinoma cells) (ATCC CCL-185) weremaintained in T-flasks at 37° C. with DMEM (GIBCO) that contained 5%fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mLstreptomycin (Invitrogen).

Mouse Bone Marrow-Derived DCs

C57BL/6 mice were used at 10-14 weeks of age and their bone marrow cellswere isolated from femurs and tibias and seeded on Costar 24-well cellculture plates in 1 mL of RPMI 1640 medium that was also supplementedwith 10% heat-inactivated FBS, 2 mM 1-glutamine, nonessential aminoacids, sodium pyruvate, HEPES (all from GIBCO), 5.5×10⁻²M 2-ME(Sigma-Aldrich), 100 units/mL penicillin, 100 μg/mL streptomycin(Invitrogen) and 15 ng/mL recombinant mouse GM-CSF (PeproTech). On Day3, 1 mL of medium that contained 10 ng/mL of GM-CSF was added to plates.On Day 5, another 0.5 mL fresh medium that contained 10 ng/mL of GM-CSFwas added. The 6- to 7-day-culture BMDCs (>80% CD11c+ cells) were used.All experiments were conducted in accordance with the guidelines ofLaboratory Animal Center of National Tsing Hua University (NTHU). Theanimal use protocols have been reviewed and approved by the NTHUInstitutional Animal Care and Use Committee (Approved protocol no.09733).

Plasmid Construction

The HA gene of A/Thailand/1(KAN-1)/2004/H5N1 (SEQ ID NO: 1) was providedby Dr. Prasert Auewarakul, Siriraj Hospital, Mahidol University,Thailand. The NA gene of A/Viet Nam/1203/2004/H5N1 (SEQ ID NO: 27) wasobtained from Academia Sinica, Taiwan. The M1 (SEQ ID NO: 21) and M2(SEQ ID NO: 23) genes of A/WSN/33/H1N1 were obtained from virus stocksusing reverse transcription-PCR. The genes of HA (A/Anhui/1/2005/H5N1),enhanced florescence protein (EGFP), flagellin (FliC), and profilin(PRO) were purchased from synthesized sequences (Mr. Gene) based on theNCBI GenBank accession numbers GU983383.1, AY649721.1 and AY937257.1,respectively. Each gene fragment was subcloned into pFastbac Dual(Invitrogen) using BamHI/NotI site for HA, XhoI/KpnI site for M1,EcoRI/HindIII site for M2, XhoI/KpnI site for NA, EcoRI/HindIII site forEGFP/M2 fusion, EcoRI/HindIII site for FliC/M2 fusion, and EcoRI/HindIIIsite for PRO/M2 fusion. These inserted vectors were then transformedinto E. coli strain DH5a and selected by ampicillin. All the insertedsequences were confirmed by DNA sequence analysis (Mission Biotech Inc.,Taipei, Taiwan).

Generation of Recombinant Baculoviruses

The pFastbac Dual plasmids encoding each specified gene(s) weretransformed into E. coli strain DH10Bac (Invitrogen) and selected on anLB plate that contained kanamycin (Invitrogen), gentamicin (Invitrogen),tetracycline (Invitrogen), Bluo-gal (Invitrogen), and IPTG (BioRad). Theselected colonies or the recombinant bacmids were confirmed by PCR usingM13 primers, then transfected into Sf9 cells using Cellfectin(Invitrogen). After 4 days, the recombinant baculoviruses were collectedfrom culture supernatants and the virus titers were determined using anID50 software.

Production and Purification of Influenza VLPs

The VLPs that were expressed by two viral proteins and Sf9 cells wereinfected with BacHA-M1 recombinant baculovirus at an MOI of 1. The VLPsthat were expressed by three viral proteins were co-infected withBacHA-M1 and Bac-NA recombinant baculoviruses at an MOI of 3 and 1,respectively. The VLPs that were expressed by four viral proteinsincluding M2 fusion proteins were co-infected with BacHA-M1 and BacM2-NA(or BacEGFP/M2-NA, BacNA-M2/FliC, BacNA-M2/PRO) recombinantbaculoviruses at an MOI of 3 and 1, respectively. At 72 hours postinfection, the culture supernatants were harvested and clarified bycentrifugation for 0.5 hour at 12,000 rpm at 4° C. Then, they wereconcentrated and pelleted for 2 hours at 33,000 rpm and 4° C. using aHitachi RPS40ST rotor. The particles were resuspended in 0.8 mL of PBSbuffer, and loaded on a 0-60% (w/v) discontinuous sucrose gradient,before being ultracentrifuged by a Hitachi RPS40ST rotor 4 hours at33,000 rpm and 4° C. Following ultracentrifugation, the fractions (0.8mL) were collected and the samples in each fraction were analyzed bySDS-PAGE and Western blotting.

Hemagglutination Titer

For the hemagglutination titer test, a series of two-fold dilutions ofinfluenza VLPs in PBS were prepared and incubated at 25° C. for 40 minwith 50 μL of 0.5% Turkey red blood cells. The extent ofhemagglutination was observed visually, and the highest dilution thatcan agglutinate red blood cells was determined.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) andWestern Blotting

Each sucrose gradient fraction sample was treated with 1×SDS gel-loadingbuffer (50 mM Tris-HCl, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenolblue, and 10% glycerol) for 5 min, resolved on 12% SDS-PAGE, and thentransferred to PVDF membranes. Following the transfer, the PVDFmembranes were blocked using 10% milk on an orbital shaker for 1 hour.Then the membranes were first reacted with anti-HA (Abcam ab21297),anti-M1 (Abcam ab25918), anti-NA (Abcam ab70759), anti-M2 (novusNB100-2073) or anti-EGFP (novus NB-600-601ss) antibodies for 1 hour,then reacted with the goat anti-rabbit or goat anti-mouse IgG conjugatedwith HRP (horse radish peroxidase) for 1 hour. Enhancedchemiluminescence (ECL) was detected through binding to HRP andvisualized on a Fuji Medical X-ray film using a Western blot detectionsystem (Amersham Bioscience).

Transmission Electron Microscopy (TEM)

The purified sucrose fractions containing VLPs were pooled andultracentrifugated using the Hitachi RPS40ST rotor 2 hours at 33,000 rpmand 4° C. to remove the sucrose and to pellet the VLPs. The VLP pelletswere resuspended with 200 μL PBS. For deep staining of the grid, 3 μLpurified VLPs was added to the carbon-coated copper grid and stainedthree times with uranyl acetate before being vacuum-dried overnight.

Confocal Fluorescence Microscopy

A549 cells were grown on glass coverslips. VLPs were labeled with DiI(Vybrant DiI cell labeling solution) and A549 cells were labeled withDiD (Vybrant DiD cell labeling solution). Labeled VLPs were incubatedwith labeled A549 cells and analyzed by confocal fluorescencemicroscopy. DiI was excited by the 561 nm line of a laser. DiD wasexcited by the 633 nm line of a laser. EGFP was excited by the 488 nmline of a laser.

Mouse Immunization

A group of five female BALB/c mice (6 to 8 weeks old) was used forimmunization studies. Immunizations were performed by intramuscularinjection of 15 μg of the purified VLPs (suspended in PBS at pH 7.4) foreach dose and three doses were conduced in a 3-week interval. Blood wascollected 2 weeks after third immunization and serum was isolated. Allexperiments were conducted in accordance with the guidelines of theLaboratory Animal Center of National Tsing Hua University (NTHU). Animaluse protocols were reviewed and approved by the NTHU InstitutionalAnimal Care and Use Committee (approval no. 09733).

H5-Pseudotyped Particles (H5Pp)

3×10⁶ HEK293T cells were transfected with pNL-Luc-E⁻R⁻, pcDNA3.1-HA(A/Thailand/1(KAN-1)/2004/H5N1 and A/Anhui/1/2005/H5N1) and pcDNA4B-NA(A/Viet Nam/1203/2004/H5N1) vectors. Cell supernatant that containedpseudotyped HIV-1 particles with H5N1 HA and NA were collected 48 hourspost-transfection and purified through a 0.45 μm filter. The supernatantwas concentrated by ultracentrifugation at 33,000 rpm for 2.5 hours, andthen each pellet was dissolved in 100 μL PBS. An HIV-1 p24 ELISA assaykit (BioChain) was used to quantify the H5pp particles.

Neutralization Assay

MDCK cells (4000 cells/well) were seeded in 100 μL of DMEM in 96-wellplates. The amount of 25 ng of p24 H5pp was incubated with two-foldserial dilutions of serum (starting dilution 1:40) for 1 hour at 37° C.in 60 μL DMEM. Then 100 μL of fresh medium was added and 140 μL of thevirus-serum mixtures was transferred to the cells. The luciferase assaywas performed 48 hours following the direct addition of neoliteluciferase substrate (PerkinElmer). The neutralization titer was definedas the reciprocal of the dilution that yielded 50% neutralizationdetermined using an ID50 software.

Analysis of Cytokine Production

DCs were untreated or individually treated with LPS 50 ng/mL from E.coli 0111:B4 (Sigma), PBS, 1 μg/mL VLP, FliC-VLP or PRO-VLP for 6 hours,with the addition of a protein transport inhibitor, brefeldin A (10μg/mL) (Biolegend), for the final 4.5 hours. Cells were then fixed andpermeabilized, and the intracellular cytokines were stained with TNF-αmAb (Biolegend). They then underwent flow cytometry (FACS Calibur, BD)and analyzed using CellQuest software (BD Biosciences).

Analysis of DC Maturation

After the BMDCs were untreated or treated with VLPs, FliC-VLPs orPRO-VLPs (5 μg/mL) for 16 hours, the cells and supernatants wereharvested and stained with monoclonal antibodies against conjugatedCD11c-FliC, conjugated CD40-PE, and conjugated CD86-PE (Biolegend). Thecells were then acquired and analyzed using flow cytometry (FACSCalibur, BD).

Results

Baculovirus-Insect Cell Expression of Influenza VLPs

A baculovirus-insect cell expression system was used to prepare theinfluenza VLPs by the over-expression of two viral proteins (HA, M1),three viral proteins (HA, NA, M1), and four viral proteins (HA, NA, M1,M2). The cDNAs of the four viral proteins were obtained from differentinfluenza virus strains: HA (A/Thailand/1(KAN-1)/2004/H5N1) (SEQ ID NO:1), NA (A/Viet Nam/1203/2004/H5N1) (SEQ ID NO: 27), M1 (A/WSN/1933/H1N1)(SEQ ID NO: 21) and M2 (A/WSN/1933/H1N1) (SEQ ID NO: 23). These geneswere cloned into the baculovirus vector under two promoters, polyhedron(pH) and p10, to generate a series of recombinant baculoviruses(BacHA-M1, BacNA, BacM2-NA) (FIGS. 11A-C). Influenza VLPs were obtainedfrom Sf9 cells that were infected with BacHA-M1 (two viral proteins),co-infected with BacHA-M1 and BacNA, or co-infected with BacHA-M1 andBacM2-NA. Influenza VLPs were obtained from the culture supernatants andpurified by ultracentrifugation and sucrose gradient sedimentation. Theformation of influenza VLPs was in the sucrose gradient fractionsverified by Western blotting in the presence of two viral proteins HAand M1 (FIG. 12A), three viral proteins HA, NA, M1 (FIG. 12B), and fourviral proteins HA, NA, M1, M2 (FIG. 12C). The TEM results reveal thatthe VLPs obtained from infected Sf9 cells were roughly spherical andwere pleomorphic. The average diameters of the influenza VLPs were 102±3nm (N=10) for two viral proteins, 100±4 nm (N=10) for three viralproteins, and 97±13 nm (N=10) for four viral proteins (FIG. 13).Distinctive influenza spike projections were observed on the surface ofthe VLPs expressed using three and four viral proteins (FIG. 13). Theinfluenza VLPs that were expressed using two, three and four viralproteins were all capable of maintaining red blood cell agglutination asdetermined from the HA titers of 512 (two viral proteins), 256 (threeviral proteins), and 512 (four viral proteins) per 50 μL.

Production of Influenza VLPs with EGFP/M2 Fusion Protein

It was proposed that M2 protein can be used as a molecular fabricator(i) without disrupting the assembly of VLPs and (ii) while retaining thenative structures of HA and NA envelope proteins on the particlesurfaces. Fabrication of influenza VLPs was obtained by theover-expression of four viral proteins by a direct fusion of M2 to EGFP.The EGFP gene was added to the N terminus of the M2 gene to constructthe baculovirus (BacEGFP/M2-NA). Sf9 cells were co-infected with tworecombinant baculoviruses (BacHA-M1 and BacEGFP/M2-NA) to generate theEGFP-VLPs. Direct fusion of EGFP to M2 did not influence the formationof VLPs as revealed by the presence of four viral proteins in thesucrose gradient fractions (FIG. 14A) and the TEM visualization of thespherical and pleomorphic particles with an average diameter of 93±13 nm(N=10) (FIGS. 14B-E).

To further show the functionality of the EGFP-VLPs, live cell imagingwas used to visualize the uptake of EGFP-VLPs in A549 cells. Usingconfocal microscopy at various wavelengths of emitted light, greenfluorescent spots of the EGFP-VLPs were observed inside the A549 cellswith light that was excited at 488 nm (FIG. 15A), and overlapped the redfluorescent spots of the VLPs that were stained with DiI, which is afluorescent lipophilic dye that was used to label viral membranes withinthe A549 cells with an excited light wavelength of 561 nm (FIG. 15B). Inparallel, A549 cells were labeled with DiD, a fluororescent lipophilicdye for labeling cell membranes, yielding blue fluorescent spots with anexcited light wavelength at 633 nm. These results reveal that influenzaVLPs can be generated by the M2 fusion of EGFP for imaging single virusentering A549 cells.

Production of Influenza VLPs with Flagellin/M2 and Profilin/M2 FusionProteins

Two molecular adjuvants, FliC and PRO, were then replaced with EGFP togenerate two molecular adjuvanted VLPs, FliC-VLPs and PRO-VLPs. Thefull-length genes of FliC and PRO were fused in front of the M2 gene toconstruct the recombinant baculoviruses, BacFliC/M2-NA and BacPRO/M2-NA.Sf9 cells were co-infected with BacHA-M1 and Bac FliC/M2-NA or BacHA-M1and BacPRO/M2-NA to yield FliC-VLPs and PRO-VLPs. Direct fusion of FliCand PRO to M2 formed FliC-VLPs (FIG. 16A) and PRO-VLPs (FIG. 17A) asevidenced by the presence of the fusion proteins and other three viralproteins HA, NA, M1 in the sucrose fractionated samples. Themorphologies of FliC-VLPs and PRO-VLPs were spherical and pleomorphic,with average diameters of 94±7 nm (N=10) and 94±13 nm (N=10),respectively (FIGS. 6B-E and 17B-E). These results reveal that themolecular adjuvanted VLPs can be obtained using M2 fusion proteins.

To study the effects of molecular adjuvanted VLPs on dendritic cells,mouse BMDCs were obtained in vitro, treated with various influenza VLPs(VLPs, FliC-VLPs, PRO-VLPs) and then analyzed using FACS analysis. Theresults indicate that the production of TNF-α in BMDCs increased from98.2% (VLP) to 148.3% (FliC-VLP) and 119.4% (PRO-VLP) than in thecontrols of untreated (10.6%) and LPS-treated BMDC cells (86.5%) (FIG.18). The maturation of BMDCs that was caused by influenza VLPs was alsoelucidated by measuring the amount of the co-stimulatory molecules ofCD40 and CD86 on the surfaces of BMDCs. The results show that since themean fluorescence intensities (MFI) of CD40⁺CD11c⁺ and CD86⁺ CD11c⁺ inBMDCs upon treatment with FliC-VLPs and PRO-VLPs increased above thosein VLPs (FIG. 19), the molecular adjuvanted VLPs (FliC-VLPs andPRO-VLPs) induced BMDCs to produce more TNF-α and to promote more DCmaturation in vitro.

To investigate whether immunization with the molecular adjuvatedFliC-VLPs and PRO-VLPs can elicit more potent immune responses than thewild-type VLPs, BALB/c mice were immunized with VLPs, FliC-VLPs, andPRO-VLPs at 15 μg (total protein) per dose for three immunizations. Themouse sera were collected one week after the third immunization andanalyzed for H5pp neutralization. The results show that the antiserathat were collected from mice that have been immunized by VLPs,FliC-VLPs and PRO-VLPs neutralized H5pp of the homologous KAN-1 strain(FIG. 20A) and the heterologous Anhui strain (FIG. 20B) were all in adose-dependent manner. For neutralization of the homologous strain, the50% neutralization titers were log₂ 6.5 for VLP antisera, log₂ 11.2 forFliC-VLP antisera, and log₂ 12.8 for PRO-VLP antisera. Forneutralization of the heterologous Anhui strain, the 50% neutralizationtiters were log₂ 5.7 for VLP antisera, log₂ 8.8 for FliC-VLP antisera,and log₂ 9.3 for PRO-VLP antisera. Immunization using the fabricatedVLPs that contained the molecular adjuvants (PRO-VLPs and FliC-VLPs)elicited more potent neutralizing antibody responses in mice against thehomologous and the heterologous H5N1 viruses than the wild-type VLPs.

What is claimed is:
 1. A DNA vaccine comprising a hyperglycosylatedmutant HA gene, wherein the hyperglycosylated mutant HA gene encodes aprotein comprising an amino acid sequence of SEQ ID NO: 4, 6, or
 10. 2.The DNA vaccine of claim 1, which elicits an immune response against aplurality of avian influenza virus subtypes in a subject.
 3. A DNAvaccine composition comprising: (a) a DNA vaccine of claim 1; and (b) abooster.
 4. The DNA vaccine composition of claim 3, wherein the boosteris an influenza virus-like particle (VLP).
 5. The DNA vaccinecomposition of claim 4, wherein the influenza virus-like particle isderived from cell infected by recombinant baculoviruses comprising oneor more plasmids containing HA gene, M1 gene, NA gene and FliC-M2 gene,which encodes FliC-M2 fusion protein.
 6. The DNA vaccine composition ofclaim 3, which further comprises an adjuvant.
 7. The DNA vaccinecomposition of claim 6, wherein the adjuvant is an aluminum-containingadjuvant.
 8. The DNA vaccine composition of claim 3, wherein the DNAvaccine and the booster have a mass ratio of 5 to
 3. 9. The DNA vaccinecomposition of claim 3, which elicits an immune response against aplurality of avian influenza virus subtypes in a subject.